EXPRESSION AND PURIFICATION OF HUMAN p62 (HIS-TEV-mCherry-p62)

Elisabeth Holzer, Gabriele Zaffagnini

Published: 2023-07-31 DOI: 10.17504/protocols.io.j8nlkob61v5r/v1

ASAPCRN

Abstract

This protocol describes how to express and purify human p62 tagged N-terminally with HIS and TEV. The expression is performed with E. coli Rosetta pLysS cells. The protein is purified via a HisTrap column and gel filtration (SEC).

Before start

Lysis Buffer:

25 mM HEPES pH 7.5

150 mM NaCl

25 mM Imidazole

2 mM MgCl2

Freshly added: 2 mM β-Mercaptoethanol, Roche Protease Inhibitor (Merck, #5056489001), DNAse I (Sigma, #DN25-1G)

HisTrap Buffer A (filtered, degassed):

25 mM HEPES pH 7.5

500 mM NaCl

25 mM Imidazole

Freshly added: 2 mM β-Mercaptoethanol

HisTrap Buffer B (filtered, degassed):

25 mM HEPES pH 7.5

500 mM NaCl

400 mM Imidazole

Freshly added: 2 mM β-Mercaptoethanol

SEC Buffer (filtered, degassed):

25 mM HEPES pH 7.5

500 mM NaCl

Freshly added: 1 mM DTT

Steps

Expression of p62

Grow E. coli Rosetta pLysS cells in 8L Luria broth medium at 37°C until an OD_{600 nm}of 0.8 is reached.

Induce protein expression with 150micromolar (µM) IPTG for 5h 0m 0s at 25°C.

Centrifuge cells at 3000 rcf, 4°C, 0h 15m 0s

Aspirate media and resuspend pellet in 10mL Lysis Buffer

Flash freeze sample in liquid nitrogen

Store at -80°C until use.

Purification of p62

Thaw sample at 37°C

Sonicate sample 5 cycles at 65% power for 0h 0m 30s

8.1.

Repeat sonication for a total of three times

Centrifuge sample 140,000 rcf at 4°C for 0h 30m 0s

9.1.

During centrifugation perform equilibration

10.

Equilibrate a HisTrap HP column (GE Healthcare) with 5 column volumes of water and 5 of HisTrap Buffer A

11.

Filter the supernatant through 0.2 µm syringe filter

12.

Load sample onto the equilibrated HisTrap column at 4°C

13.

Elute protein via a stepwise imidazole gradient using HisTrap Buffers A and B and elution steps: 25 mM, 62.5 mM, 118.75 mM, 148.75 mM, 212.mM, 306.25 mM and 400 mM imidazole

14.

Add 100µg to 150µg TEV protease to pooled protein-containing fractions and incubate 0h 0m 30s at 4°C to cleave.

15.

Concentrate resulting protein with a 30 kDa MWCO concentrator (Millipore) to between .5mL and 1mL

16.

Apply protein to a Superdex 200 Increase 10/300 column (Cytiva) pre-equilibrated with SEC Buffer.

17.

Freeze purified protein in liquid nitrogen and store at -80°C

18.

Protein purity can be determined by SDS PAGE analysis